Abstract:

The invention provides methods for expanding mesenchymal stem cells (MSCs)
in non-adherent cultures. The methods include the propagation of MSCs in
or on non-adherent matrices. The invention further provides
administration and the use of cells propagated by the method of the
invention for administration and preparation of a therapeutic agent. The
invention further provides kits including cells propagated by the methods
of the inventions.

Claims:

1. A method for propagation of a non-adherent culture of mesenchymal stem
cells (MSCS) comprising expanding MSCs in or on a non-adherent matrix.

2. The method of claim 1, comprising encapsulation of MSCs in Matrigel®
or Hydrogel.

3. The method of claim 1, comprising the cells propagated on agarose or on
Teflon®.

4. The methods of claim 1, wherein the cells are propagated in the
non-adherent culture without the use of trypsin.

5. The methods of claim 1, comprising mechanical manipulation of the MSCs.

6. The method of claim 1, further comprising a biological sample
containing MSCs.

7. The method of claim 6, further comprising isolating the MSCs from the
biological sample containing the MSCs.

8. The method of claim 7, wherein the isolated MSCs are substantially
purified.

[0003]This invention relates to methods of growth of mesenchymal cells
under non-adherent conditions. The method allows for expansion of
mesenchymal cells in suspension for research or therapeutic uses.

BACKGROUND OF THE INVENTION

[0004]Mesenchymal stem cells are the formative pluripotential blast cells
found inter alia in bone marrow, blood, dermis, and periosteum that are
capable of differentiating into any of the specific types of mesenchymal
or connective tissues (i.e. the tissues of the body that support the
specialized elements; particularly adipose, osseous, cartilaginous,
elastic, and fibrous connective tissues) depending upon various
influences from bioactive factors, such as cytokines. In contrast to
their hematopoietic counterparts, MSCs are adherent and can be expanded
in culture. A number of U.S. Patents, e.g., U.S. Pat. Nos. 5,486,359;
5,591,625; 5,736,396; 5,811,094; 5,827,740; 5,837,539; 5,908,782;
5,908,784; 5,942,225; 5,965,436; 6,010,696; 6,022,540; 6,087,113;
5,858,390; 5,804,446; 5,846,796; 5,654,186; 6,054,121; 5,827,735; and
5,906,934 (all of which are incorporated herein by reference) disclose
mesenchymal stem cells (MSC), which can be differentiated into several
progenitor cells, for example muscle progenitor cells, connective tissue
cell progenitor cells, or hepatic oval cells. Muscle progenitor cells
differentiate further into cardiac, skeletal, and smooth muscle cells,
whereas the connective tissue cell progenitor may differentiate into
bone. The patents above further teach transgenic MSCs that carry a
transgene, methods to promote differentiation of MSCs along specific
paths, and therapeutic methods including the use of MSCs.

[0005]Human MSC (hMSC) can be identified by the presence or absence of
specific cell surface markers (Pittenger and Martin, Circ. Res. 95:9-20,
2004, incorporated herein by reference). Typically, hMSC can be
identified by the presence of surface markers CD13, CD29, CD44, CD49a, b,
c, e, f, CD51, CD54, CD58, CD71, CD73, CD90, CD102, CD105, CD106, CDw11,
CD120a, CD120b, CD123, CD124, CD126, CDC127, CD140a, CD166, P75, TGFb1R,
TGFbIIR, HLA-A, B, C, SSEA-3, SSEA-4, D7; and the absence of surface
markers CD3, CD4, CD6, CD9, CD10, CD11a, CD14, CD15, CD18, CD21, CD25,
CD31, CD34, CD36, CD38, CD45, CD49d, CD50, CD62E, L, S, CD80, CD86, CD95,
CD117, CD133, SSEA-1. Monoclonal antibodies specific to MSCs have also
been identified (e.g., U.S. Pat. Nos. 5,486,359 and 5,811,094). However,
most surface markers have been found inadequate as a means to identify
stem cells because putative marker(s) may also be found on nonstem cells,
or a particular marker may only be expressed on a stem cell at a certain
stage or under certain conditions, such as CD34 on hematopoietic stem
cells. Nevertheless, surface markers and other attributes are useful in
characterizing a stem cell as isolated or cultured, to detect changes in
cells in culture over time, and as a means to begin to understand its
potential interactions with neighboring cells and the cell environment
(Pittenger and Martin, Circ. Res. 95:9-20, 2004).

[0006]Mesenchymal stem cells can be isolated from a number of cells and
tissues including bone marrow, embryonic yolk sac, placenta, umbilical
cord, fetal and adolescent skin, and blood, and propagated in culture.
Friedenstein et al. (Exp. Hematol. 4:267-274, 1976, incorporated herein
by reference) initially isolated MSCs by their adherence to tissue
culture surfaces. Similar methods for isolation of MSCs are still
commonly used.

[0007]Plating studies indicate that MSCs are present at as a rare
population of cells in bone marrow, representing about 0.001-0.01% of
nucleated cells. However, MSCs can be readily expanded when grown at a
very low plating density. Cotler et al. (Proc. Natl. Acad. Sci. USA.
97:3213-3218) noted that the number of colonies formed per 100 cells
plated remained constant when the density of plating was varied from 0.5
to 12 cells per cm2. However, the size of the colonies decreased
markedly when the cells were plated at higher densities. Colonies of
maximal size were obtained when cells were plated at 1.5 to 3.0 cells per
cm2. Plating at such low densities requires the use of large amount
of tissue culture dishes, reagents, and space. Methods for culturing of
MSCs in a less resource intensive manner is desirable.

[0008]Adult bone marrow-derived MSCs engraft in numerous organs and
differentiate along tissue-specific lineages when transplanted into
animals. They migrate into areas of muscle degeneration to undergo
myogenic differentiation in immunodeficient mice. Injection of MSCs
directly into infracted swine heart has been shown to induce myocardial
regeneration and improved cardiac function (Shake et al., Ann. Thorac.
Surg. 73:1919-1925, 2002). In addition, MSCs implantation has been
demonstrated to induce therapeutic angiogenesis in a rat model of
hindlimb ischemia through vascular endothelial growth factor (VEGF)
production by MSCs (Al-Khaldi et al., Gene Ther. 10:621-629, 2003). In
humans, bone marrow-derived MSCs have been used to regenerate the marrow
microenvironment after myeloablative therapy. When introduced into the
infracted heart, MSCs prevent deleterious modeling and improve recovery.
Interestingly, implanted cells do not appear to expand after implantation
when engrafted to tissue other than bone. Experiments using MSCs labeled
with membrane dyes that would be diluted out after about 3 cell divisions
were found months later even in repairing tissue (Pittenger and Martin,
Circ. Res. 95:9-20, 2004).

[0009]Clinical trials have been initiated in several countries to test
cell-based therapies for the treatment of the injured heart. However, no
studies have demonstrated incorporation of MSCs into regenerating tissue.
It has been suggested that the MSCs exert a therapeutic effect by
paracrine actions exerted by the cells through the release of soluble
factors (See e.g., Gnecchi et al., FASEB J. 20:661-669, 2006; and Nagaya
et al., Circulation. 112:1128-1135, 2005). This theory is supported by
data therein demonstrating that conditioned media from transgenic MSCs
overexpressing the prosurvival gene Akt limits hypoxia-induced apoptosis
and triggers vigorous spontaneous contraction of adult rat cardiomyocytes
in culture. Moreover, injection of concentrated conditioned media from
the Akt transgenic MSCs into infracted rat hearts significantly limited
infarct size and improved ventricular function relative to controls
(Gnecchi et al., 2006).

[0010]Studies have demonstrated that upon transplantation of cells into
cardiac tissue (e.g., by injection) less than 3% of injected MSCs persist
after 2 weeks (Mazhari & Hare, Nature Clinical Practice Cardiovascular
Medicine 4: suppl 1; S21-S26, 2007). This may be due to the adherent
culture methods used to culture the MSCs. MSCs in bone marrow are able to
adhere to bone to allow for proliferation. No comparable surface is
present in muscle or many other tissues in which MSCs have been
demonstrated to be beneficial. Current culture methods select for cells
that are able to adhere to culture dishes through repeated rounds of
trypsinization. When transplanted into cardiac tissue for example, MSCs
may fail to proliferate due to their inability to adhere to a cardiac
tissue surface, minimizing the contribution of MSCs to regenerating
tissue.

[0011]Methods of culture of MSCs that do not include adherence to a
surface and/or reduce the need for multiple rounds of trypsinization for
propagation of cells may improve the effects of MSC at sites of injury,
for example, by providing cells that are more able to proliferate at the
site of injury.

SUMMARY OF THE INVENTION

[0012]The invention provides methods for the propagation of mesenchymal
stem cells (MSCs) in non-adherent culture, eliminating the need for
trypsinization in propagation of MSCs.

[0013]Accordingly, an aspect of the invention features a method for
culturing MSCs under non-adherent conditions in or on a non-adherent
matrix to obtain an expanded population of MSCs. The methods include
formation of MSC spheres (MSCS) in or on several different non-adherent
matrices, including incorporation of cells into biocompatible matrices
such as Hydrogel and Matrigel®; culture of cells on or between layers
of agarose; and culture of cells in Teflon® bags. After isolation of
MSCs from a sample, the cells are propagated without treatment with
trypsin after initial cell selection. MSCS are optionally mechanically
manipulated, collected by centrifugation, and resuspended in fresh media
for continued propagation, or resuspended in an appropriate buffer for
administration to a subject.

[0014]An aspect of the invention features a method for therapeutic
administration to a subject in need of treatment with MSCs comprising;
i)obtaining MSCs, for example by isolating the cells from a sample, ii)
culturing the cells in a non-adherent manner to generate an expanded
population of cells, and iii) administering the cells to the subject. In
an embodiment, the MSCs are administered to an individual having a
condition or disease susceptible to treatment with MSCs

[0015]An aspect of the invention provides for the use of MSCs cultured
under non-adherent conditions for use as a medicament for the treatment
of a condition or disease susceptible to treatment with MSCs.

[0016]An aspect of the invention includes kits containing MSCs expanded
under non-adherent conditions in appropriate packing material. In an
embodiment, the kits further include reagents or materials for
propagation of the cells under adherent and/or non-adherent conditions.

[0017]In some embodiments of the invention, the methods further include
obtaining a sample that contains MSCs, and may further include isolating
the MSCs to obtain a substantially purified sample of MSCs.

[0018]In some embodiments of the invention, culturing the MSCs increases
the expansion of the cells by at least 2 fold, preferably at least 10
fold or 100 fold, more preferably 1000 fold, 10,000 fold, or 100,000
fold. In another embodiment of the first or second aspects of the
invention, the MSCs are maintained in non-adherent culture for at least
one week, preferably at least two weeks, at least a month, or at least
two months.

[0019]In some embodiments of the invention, the cultured MSCs are suitable
for administration to a subject, preferably a human subject.

[0020]In some embodiments of the invention, the MSCs are allogenic or
autologous to the subject to whom the cells are administered.

[0021]In an embodiment, the MSCs may express classic surface markers
including CD105, CD73 and CD90 but lack expression of CD34 or CD45.

Definitions

[0022]By "administering", "therapeutic administration" and the like is
meant providing to a human patient a pharmaceutical preparation
containing the MSCs, optionally in the form of MSC spheres or foci, or
their progeny or derivatives in a suitable formulation. The preferred
method of administration can vary depending on various factors, e.g., the
components of the pharmaceutical preparation, site of the potential or
actual disease, and severity of disease.

[0023]By "allogenic" is meant involving, derived from, or being
individuals of the same species that are sufficiently unlike genetically
to interact antigenically.

[0024]By "animal" is meant to be preferably a mammal. A mama can be human
or non-human including, but not limited to laboratory and/or commercially
important mammals, such as mouse, rat, rabbit, monkey, dog, cat, pig,
cow, sheep, and goat.

[0025]By "autologous" is meant derived from the same individual or
involving one individual as both donor and recipient.

[0026]By "cell culture" is meant grown outside of the body in a dish,
flask, or other container in the presence of growth media Cell culture
can be performed with transformed or immortalized cell lines. Cell
culture can also be performed with "primary cells" removed from an
animal, such as a mammal, and are not transformed or immortalized.
Primary cells can be dividing or non-dividing cells. For example, the
cells can be bone marrow cells, umbilical cord blood cells, or
mesenchymal stem cells.

[0027]By a "condition or disease susceptible to treatment with MSCs" is
meant a malady that has been demonstrated to be treated using MSCs, for
example muscle disease, neural disease, and vascular disease. Theses
diseases have been demonstrated to be susceptible to treatment with MSCs.
For example, demonstrated therapeutic effects include those shown in U.S.
Pat. No. 5,811,094 to promote connective tissue regeneration; U.S. Pat.
No. 5,858,930 for repair of skin and soft tissue defects; U.S. Pat. No.
6,387,369 for cardiac muscle regeneration; U.S. Pat. No. 6,875,430 for
treatment of immune responses in transplantation; U.S. Pat. No. 7,029,666
for muscle and connective tissue repair, U.S. Pat. No. 7,097,832 for
enhancing blood vessel formation; and U.S. Pat. No. 7,160,724 for repair
of the brain and spinal cord.

[0028]By "effective amount" is an amount sufficient to effect beneficial
or desired clinical or biochemical results. An effective amount can be
administered one or more times. For purposes of this invention, an
effective amount is the amount of MSCs to effect beneficial engraftment
of the cells.

[0029]By "engraftment" is meant the implantation of cells in the body,
and/or replacement of lost or damaged cells with injected cells. The
engrafted cells persist in a particular location over time following
transplantation of the cells into a mammal (e.g., a human).

[0030]By the term "expanded population" is meant a population of cells,
e.g., MSCs isolated from bone marrow or other tissue, wherein at least
50% of the cells have divided at least once.

[0031]A molecule is a "marker" of a desired cell type if it is found on a
sufficiently high percentage of cells of the desired cell type, and found
on a sufficiently low percentage of cells of an undesired cell type, such
that one can achieve a desired level of purification of the desired cell
type from a population of cells comprising both desired and undesired
cell types by selecting for cells in the population of cells that have
the marker. A marker can be displayed on, for example, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the
desired cell type, and can be displayed on fewer than 50%, 45%, 40%, 35%,
30%, 25%, 20%, 15%, 10%, 5%, 1% or fewer of an undesired cell type. It is
preferred that a marker be displayed on 90% or more of a desired cell
type, or on fewer than 10% of a desired cell type.

[0032]A desired cell type is negative for a cell surface-expressed marker
or lacks expression of the marker if fewer than 50 marker molecules per
cell are present on the cell surface of the desired cell type. Techniques
for detecting cell surface-expressed marker molecules are well known in
the art and include, e.g., flow cytometry. One skilled in the art can
also use enzymatic amplification staining techniques in conjunction with
flow cytometry to distinguish between cells expressing a low number of a
marker molecule and cells that do not express the marker molecule (see,
e.g., Kaplan, Front. Biosci. 7:c33-c43, 2002; Kaplan et al., Amer. J.
Clin. Pathol. 116:429-436, 2001; and Zola et al., J. Immunol. Methods
135:247-255, 1990).

[0033]By "non-adherent matrix" is meant a material which cells can grow
in, or on a material that prevents adhesion to a cell culture container
surface. For example, growing cells in a non-adherent matrix (e.g.,
Hydrogel, BD Biosciences or Matrigel®, BD Biosciences) can prevent
attachment to a cell culture container surface. MSCs may adopt their
typical fibroblast-like shape on the matrices, but do not attach to the
plastic culture surface. Alternatively a non-adherent matrix can be
understood to be a matrix that the cells can grow on, but do not attach
tightly to (e.g., agarose, or Teflon®). With such matrices, the MSCs
retain a rounded, rather than fibroblast shape which they obtain when
grown on plastic. In a preferred embodiment, the non-adherent matrix is
preferably biocompatible such that it can be administered to a subject
for transplant without -separation from the matrix. Alternatively, the
matrix can be of a size, shape, and resiliency that readily allows for
removal of the cells from the matrix (e.g., Teflon®) to allow the
cells to be administered to a subject.

[0034]By "mesenchymal stem cell" (MSC) is meant an adherent stroma cell,
for example from a biological sample such as bone marrow or umbilical
cord blood, isolated by methods such as those provided herein and by U.S.
Pat. Nos. 5,486,359; 5,654,186; 5,827,735; 5,858,390; 5,906,934;
5,908,784; 5,965,436; and 7,060,494. Such cells have been characterized
by being multipotent stem cells that have the capacity to differentiate
into osteoblasts, adipocytes and chondrocytes in vitro and express the
surface antigens CD105, CD73 and CD90, but not CD45 or CD34 (Dominici et
al, Cytotherapy 8:315-317, 2007)

[0035]By a "muscle cell" is meant a skeletal, smooth, or cardiac cell.

[0038]By "neural disease" is meant a disease or disorder that affects or
involves the central or peripheral nervous system. Examples of neural
diseases include multi-infarct dementia (MID), vascular dementia,
cerebrovascular injury, Alzheimer's disease (AD), neurofibromatosis,
Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis,
stroke, Parkinson's disease (PD), pathologies of the developing nervous
system, pathologies of the aging nervous system, and trauma, e.g., head
trauma Other examples of neural diseases are those that affect tissues of
the eye, e.g., the optic stalk, retinal layer, and lens of the eye, and
the inner ear. In certain embodiments, the patient may have suffered a
neurodegenerative disease, a traumatic injury, a neurotoxic injury,
ischemia, a developmental disorder, a disorder affecting vision, an
injury or disease of the spinal cord, or a demyelinating disease.

[0039]By "non-adherent culture" is meant herein as a method of propagation
of cells in vitro as in a container in the presence of growth media in a
manner in which the cells do not attach to the surface of the container
such that a substantial portion of the cells can be removed from the
surface of the container by mechanical manipulations that do not cause
significant damage to the cells. It is understood that the cells can
still be retained in or on a non-adherent matrix (e.g., on Hydrogel
spheres) and be removed from the surface of the container. Such
manipulations include, for example, gentle agitation, massage, or manual
manipulation of the container, or rinsing the container with growth media
As used herein, a substantial portion of the cells to be removed is at
least 70%, preferably at least 75%, 80% or 85%, more preferably at least
90% or 95%. Manipulations that cause damage to the cells can be
identified by determining the viability of the cells before and after
manipulation, for example by trypan blue staining. Mechanical
manipulations should cause damage to less than 20%, preferably less than
15%, or 10%, more preferably less than 5%, 2%, or 1% of the cells.

[0040]By "obtaining" as in "obtaining an agent" or "obtaining a cell"
refers to purchasing, synthesizing, or otherwise procuring an agent or
cell. Cells can be obtained, for example, from an animal including human
and non-human animals. Cells can also be obtained from cell and tissue
repositories.

[0041]By "prevent," "preventing," "prevention," "prophylactic treatment"
and the like is meant reducing the probability of developing a disorder
or condition in a subject, who does not have, but is at risk of or
susceptible to developing a disorder or condition. Prevention or
prophylactic treatment can require administration of more than one dose
of the compositions of the invention.

[0042]By "propagate", "passage", and the like is meant increasing the
volume of a cell culture and/or decreasing the amount of cells in a
specific culture volume by diluting cells in at least some fresh growth
media to allow for maintenance and/or expansion of the cell population.

[0043]By "sample" or "biological sample" is meant any biological sample
obtained from an individual, body fluid, cell line, tissue culture, or
other source.

[0044]By "stem cell" or "pluripotent stem cell," which can be used
interchangeably, is meant a cell having the ability to give rise to two
or more cell types of an organism.

[0045]By "subject" is meant a vertebrate, preferably a mammal, more
preferably a human.

[0046]By "substantially purified" is meant that the desired cells (e.g.,
MSCs) are enriched by at least 30%, more preferably by at least 50%, even
more preferably by at least 75%, and most preferably by at least 90% or
even 95%.

[0047]By "transgene" is meant any piece of a nucleic acid molecule (for
example, DNA) that is inserted by artifice into a cell transiently or
permanently, and becomes part of the organism if integrated into the
genome or maintained extrachromosomally. Such a transgene may include a
gene that is partly or entirely heterologous (foreign) to the transgenic
organism, or may represent a gene homologous to an endogenous gene of the
organism. The transgene may be introduced into the organism from which
the MSCs are isolated. Alternatively, the transgene may be introduced
using viral vectors, such as retroviral vectors (See, e.g., Gnecchi et
al., 2006).

[0048]By "transgenic cell" is meant a cell containing a transgene. For
example, a cell transformed with an expression vector operably linked to
a heterologous nucleic acid molecule can be used to produce a population
of cells having altered phenotypic characteristics. A cell derived from a
transgenic organism is also a transgenic cell so long as the cell contain
the transgene.

[0049]By "transplant" or "transplanting" is meant administering one or
more cells (or parts thereof), cell products, tissue, or cell culture
products derived from cells that are grafted into a human host. For
example, a transplant can include an MSC transplant.

[0050]By "treatment" is meant an approach for obtaining beneficial or
desired clinical results. For the purposes of this invention, beneficial
or desired clinical results include, but are not limited to, alleviation
of symptoms, diminishment of extent of disease, stabilization (i.e., not
worsening) of a state of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or undetectable.
"Treatment" can also mean prolonging survival as compared to expected
survival if not receiving treatment. "Treatment" refers to both
therapeutic treatment and prophylactic or preventative measures. Those in
need of treatment include those already with the disorder as well as
those in which the disorder is to be prevented. "Palliating" a disease
means that the extent and/or undesirable clinical manifestations of a
disease state are lessened and/or the time course of the progression is
slowed or lengthened, as compared to a situation without treatment.
Typically, the "treatment" entails administering an effective dose of
MSCs to the patient to regenerate tissue.

[0051]By a "vascular cell" is meant an endothelial cell. Endothelial cells
line the blood and lymph vessels and are present in and play a key role
in the development of organs, such as the brain, heart, liver, pancreas,
lungs, spleen, stomach, intestines, and kidneys.

[0056]FIGS. 1A-1B are images of MSC harvested from plastic adherent
culture of MSC by trypsinization and then cultured for 1 week in (A)
plastic tissue culture dish, (magnification 100×), or (B) grown in
a double layer agarose culture (magnification 100×), or cultured
for 2 weeks (C) in liquid culture above a single layer of agarose to
prevent adherence to plastic (magnification 100×)

[0057]FIG. 2A-2B is an image of MSC spheres generated in culture of MSCs
in Teflon® bags (A) grown in culture for 2 weeks (mag 100×) and
(B) for 6 weeks (mag 100×).

[0058]FIG. 3 is an image of proliferating MSCs in hydrogel for 2 weeks
(mag 100×)

[0059]FIG. 4 is an image of MSCs in a tissue culture flask after seven
passages in Teflon® bags and then transferred to a plastic culture
flask. The MSC spheres adhered to the surface of the flask within 2 to 3
days and obtained a morphology essentially identical to that observed in
cells passaged in adherent cultures (mag 100×).

DETAILED DESCRIPTION

[0060]Mesenchymal stem cells have been demonstrated to be useful in the
therapeutic methods for the repair and regeneration of tissue, especially
muscle tissue, including cardiac tissue. This is somewhat surprising as
MSCs have been demonstrated to be quiescent after injection, have low
engraftment into tissue other than bone, and to have a very low
persistence after injection.

[0061]Mesenchymal stem cells are adherent cells, and can be selected for
growth in culture by their ability to adhere to tissue culture containers
(i.e., plastic). In culture, cells are propagated by repeated rounds of
trypsinization and replating, effectively selecting for cells that are
adherent. The observed low level of engraftment and cell division in vivo
may be due to the in vitro methods of propagation of the MSCs in adherent
cultures, as no comparable surfaces are available in vivo, for example in
muscle, vascular, and neural cells.

[0062]The invention provides methods for mesenchymal stem cells (MSCs)
growth in non-adherent culture, eliminating the need for trypsinization
in propagation of MSCs. The non-adherent culture methods of the invention
allow for the propagation of MSCs that may more readily engraft into
recipient tissue and be more viable for longer periods after transplant
as they do not require a surface to which they can adhere to divide.

[0063]The non-adherent culture methods of the invention also allow for
propagation of cells in a less resource intensive manner by allowing the
cells to be grown in larger numbers in the same culture container area as
the cells do not need to all grow in the same plane of the culture
container as with adherent cells.

[0064]The invention provides culture methods that enable the generation of
MSC in non-adherent foci in various support matricies. MSCs grown under
these conditions can be passaged without trypsinization. Methods include
growth of cells encapsulated in matrices such as Hydrogel and
Matrigel®, on or between layers of agarose, or in Teflon® bags.
Cells can grow in contact with the non-adherent matrices, but do not
adhere to plastic culture containers. The lack of adherence to a surface
is notable in the MSCs grown on agarose or in Teflon® bags as can be
determined by the maintenance of their rounded shape. MSCs grown in
adherent cultures on plastic adopt an elongated, fibroblastic shape (see,
e.g., compare FIG. 1A with FIGS. 1B-1C and 2A-B).

[0065]Mesenchymal stem cells have been cultured for up to 10 passages and
can be subcultured without the need of treatment with trypsin. The
non-adherent cells express similar surface markers as cells grown under
adherent conditions (e.g., CD105), and they maintain their ability to
differentiate into multiple cell types. Optimal growth of the cells is
stimulated by basic fibroblast growth factor (bFGF) and other growth
factors including stem cell factor (SCF) and vascular endothelial growth
factor (VEGF).

[0066]Growth of non-adherent MSCs in Teflon® bags provides an
additional advantage for translation into therapeutic applications as the
MSCs can be cultured by massaging the bag to detach the cells from the
surface. When the MSCs are detached the can be maintained as MSC spheres
by regular massaging of the bag and inversion of the bag for continued
incubation. Performance of this manipulation about twice daily allows for
the MSC spheres to increase in size, and for the MSCs to continue to
proliferate and expand. The cells can readily be removed from the culture
media by centrifugation and resuspension into an appropriate buffer for
injection (e.g., phosphate buffered saline (PBS), physiological saline
solution) without the need to remove the cells from a less sturdy
non-adherent surface (e.g., Matrigel® or agarose) and without the use
of trypsin which would need to be removed from the cells prior to
administration.

[0067]It is understood that the initial source of and method of isolation
of the MSCs to be grown by the culture methods of the invention is not a
limitation of the invention. A number of methods of isolation of MSCs are
known to those skilled in the art including, but not limited to, those
set forth in U.S. Pat. Nos. 5,486,359; 5,654,186; 5,827,735; 5,858,390;
5,906,934; 5.908,784; 5,965,436; and 7,060,494.

[0068]It is further understood that the methods provided herein can be
used to culture both wild-type and transgenic MSCs such as those taught,
for example in U.S. Pat. No. 5,591,625 or in Gnecchi et al. (both
incorporated herein by reference). Transgenic MSCs can be isolated from
transgenic animals or can be transduced using vectors, including viral
vectors, for the insertion of expression constructs into the cells.

[0069]Mesenchymal stem cells cultured by the methods of the invention can
be used for any of a number of research or therapeutic purposes. For
example, a number of therapeutic methods using MSCs are known, such as
those taught in U.S. Pat. No. 5,811,094 for connective tissue
regeneration; U.S. Pat. No. 5,858,930 for repair of skin and soft tissue
defects; U.S. Pat. No. 6,387,369 for cardiac muscle regeneration; U.S.
Pat. No. 6,875,430 for treatment of immune responses in transplantation;
U.S. Pat. No. 7,029,666 for muscle and connective tissue repair; U.S.
Pat. No. 7,097,832 for enhancing blood vessel formation; and U.S. Pat.
No. 7,160,724 for repair of the brain and spinal cord (all of which are
incorporated herein by reference).

[0070]Mesenchymal stem cells cultured by the methods of the invention can
be used for the generation of cultured media to promote the growth of
cells, for therapeutic uses, or for research purposes to identify
secreted growth factors that may be responsible for the beneficial
therapeutic effects provided by MSCs.

[0071]Mesenchymal stem cells cultured by methods of the invention can be
incorporated into a kit including the cells in a container with
appropriate packing material. The kit can further contain reagents and/or
materials for culturing MSCs in adherent and/or non-adherent manner(s).

Example 1

Isolation of MSCs from Human Bone Marrow

[0072]Human bone marrow cells were obtained from normal donors following
informed consent under an Institutional Review Board approved protocol.
The mononuclear cell fraction of the bone marrow was isolated on a Ficoll
gradient and plated in a T150 Corning (Acton, Mass.) tissue culture flask
at 1-5×106 cells/ml in α-MEM media containing 20% fetal
calf serum (FCS). The cells were incubated in a humidified environment at
5% CO2 at 37° C. The media was changed weekly. Adherent cells
were grown in culture and passaged using trypsin when confluent.

Example 2

Culture of MSCs in Hydrogel

[0073]MSCs were isolated and propagated as set forth above. MSCs were
collected from adherent, confluent cultures using trypsin and
encapsulated in Hydrogel (Becton Dickson) following the manufacturer's
instructions. The encapsulated MSCs were cultured in α-MEM+20% FCS
in T75 culture flasks. At regular intervals, the non-adherent cells were
passaged by removing the supernatant, centrifuging the Hydrogel/MCS
mixture, and resuspending the cells in growth media. As shown in FIG. 3,
MSCs encapsulated in the Hydrogel proliferated and maintained a
fibroblast-like morphology. Cells encapsulated in Matrigel® gave
comparable results.

Example 3

Culture of MSCs in Agarose

[0074]Single layer agarose cultures were established in 100 mm culture
dishes on preformed layers of 0.5% agarose for double layer, and 1%
agarose for single layer agarose in α-MEM+30% FCS. MSCs were
harvested from confluent cell cultures by trypsinization and resuspended
in α-MEM+20% FCS. The MSCs were added in 10 ml of α-MEM+20%
FCS above the agarose layer. The non-adherent cells were passaged by
removing the supernatant from the agarose underlay. The cells were
centrifuged and the supernatant discarded. The cells were resuspended in
fresh media and replated over the agarose underlay. Double layer agarose
cultures were generated by incorporating the cells into a top agarose
layer (0.66%).

[0075]FIG. 1B shows cells cultured in a double layer agarose culture in
the top agarose layer. The MSCs could be visualized as single, round
cells. No proliferation was observed. However, when the cells were plated
in a liquid phase in α-MEM+20% FCS on a lower layer of 1% agarose
to prevent adherence, the MSC formed spheres and proliferated as shown in
FIG. 1C. Cells were passaged multiple times.

Example 4

Culture of MSCs in Teflon®Bags

[0076]MSCs were harvested from confluent cell cultures by trypsinization
and resuspended in 50 ml of α-MEM+20% FCS. The cells were placed in
100 ml Teflon® bags (American Fluoroceal Corp, Gaithersburg, Md.) and
cultured. At weekly intervals the bags were harvested, the cells were
centrifuged, resuspended in fresh media and placed into new Teflon®
bags.

[0077]MSCs can be cultured by massaging the bag to detach the cells from
the surface. When the MSCs are detached the can be maintained as MSC
spheres by regular massaging of the bag and inversion of the bag for
continued incubation. Performance of this manipulation twice daily allows
for the MSC spheres to increase in size, and for the MSCs to continue to
proliferate and expand (see, FIG. 2).

[0078]The culture methods have been replicated beginning with bone marrow
harvested from pig. Non-adherent cultures of pig MSC have now been
generated for animal studies. One hundred million non-adherent pig MSCs
were generated after 3 weeks of culture in Teflon® bags.

Example 5

Adherence of Cells to Plastic After Culture Under Non-Adherent Conditions

[0079]Culturing of cells under non-adherent conditions does not alter the
ability of the MSCs to adhere when provided with an appropriate
substrate. FIG. 4 shows cells grown in a tissue culture flasks after
seven passages in Teflon® bags. The morphology of the cells appears
to be identical to that of MSCs propagated continuously in adherent
culture.

Transplantation of Non-Adherent MSCs for the Treatment of Cardiac
Infarction

[0081]MSCs are isolated from rat bone marrow by standard Ficoll gradient
followed by adherent culture methods. After expansion of the cells, the
culture is split. A portion of the cells are maintained in adherent
culture, and a portion of the cells are transferred to Teflon® bags
for propagation. Cells in Teflon® bags are manipulated twice daily to
promote growth of MSC spheres, and media is changed as needed. Adherent
cells are propagated using trypsin as needed. Cells can include a marker
such as GFP or beta-galactosidase to facilitate identification of the
transplanted cells at the end of the experiment. Cells are collected and
resuspended in an appropriate buffer for administration (e.g., normal
saline).

[0082]Age and sex matched laboratory rats of a single type are divided
into four groups, sham myocardial infarction (MI), adherent MSC treated,
non-adherent MSC treated, and normal saline. In all but the sham MI
group, ligation of the left coronary artery is performed using well known
methods (see, e.g., Gnecchi et al). Briefly, animals are anesthetized and
a left thoracotamy is performed under artificial respiration. The heart
is accessed through the intercostal space, the pericardial sac is cut,
and the heart is exteriorized through the space. The left coronary artery
is legated with a silk suture about midway between the left atrium and
the apex of the heart and EKG is recorded to confirm the presence of
infarction. In sham operated animals, the artery is not legated. One hour
after infarction, an equal number of adherent or non-adherent MSCs are
injected into a total of five sites per infarct area. Normal saline is
injected into the infarct area in the control animals.

[0083]Cardiac function is analyzed at regular intervals after the surgery
and administration of the cells, for example by EKG. Either throughout
the course of the experiment, or at the end of the experiment, rats are
euthanized and hearts are excised. Analysis is performed to determine any
of a number of outcomes including, but not limited to, infarct area,
engraftment of MSCs into the infarct area, angiogenesis in the infarct
area, and/or mRNA or protein expression. Methods for performing such
analyses are known to those skilled in the art. The therapeutic effect of
the cells grown in adherent culture and non-adherent culture are compared
to each other and to control animals.

[0084]It is understood that comparable experiments can be performed using
different animals including, for example, pigs.

[0085]The invention may be embodied in other specific forms without
departing from the spirit or essential characteristics thereof The
foregoing embodiments are therefore to be considered in all respects
illustrative rather than limiting on the invention described herein.
Scope of the invention is thus indicated by the appended claims rather
than by the foregoing description, and all changes which come within the
meaning and range of equivalency of the claims are therefore intended to
be embraced therein.